Strand-selective base editing of human mitochondrial DNA using mitoBEs.

Area of Science:

• Molecular Biology
• Genetics
• Biotechnology

Background:

• Mitochondrial diseases stem from point mutations, potentially targetable by base editing.
• Delivering CRISPR guide RNAs into mitochondria for gene editing remains a significant challenge.

Purpose of the Study:

• To develop a novel base editing system for precise mitochondrial DNA modification.
• To overcome the challenge of delivering gene editing tools into mitochondria.

Main Methods:

• Engineered mitochondrial DNA base editors (mitoBEs) by fusing TALE proteins with nickases and deaminases.
• Utilized mitochondria-localized TALE binding proteins with specific nickases (MutH or Nt.BspD6I(C)) and deaminases (TadA8e or ABOBEC1/UGI).
• Delivered mitoBEs encoded in circular RNAs into patient-derived cells.

Main Results:

• Achieved precise A-to-G and C-to-T base editing in mitochondrial DNA with up to 77% efficiency and high specificity.
• Demonstrated DNA strand-selective editing, favoring retention on the non-nicked strand.
• Successfully corrected pathogenic mitochondrial DNA mutations in patient-derived cells.

Conclusions:

• mitoBEs provide a precise and efficient tool for mitochondrial DNA editing.
• Circular RNA delivery of mitoBEs is effective in correcting disease-causing mutations in patient cells.
• mitoBEs hold broad therapeutic potential for genetic mitochondrial diseases.